Nanoparticles, process for preparation and use thereof as carrier for amphipatic and hydrophobic molecules in fields of medicine including cancer treatment and food related compounds

ABSTRACT

The present invention regards nanoparticles comprising a sterol and a component derived from Quillaja saponaria Molina selected from quillaja acid and quillaja saponin, which nanoparticles do not comprise a phospholipid. It also relates to a composition comprising the nanoparticles, and the use thereof as adjuvant, especially in vaccines, as carriers for amphipathic or hydrophobic molecules and as agents for treatment of cancer. Further, it regards a method for producing the phospholipid-free nanoparticles, a method for the treatment of cancer and a method for assessing the applicability of the cancer treating method.

FIELD OF INVENTION

The present invention regards nanoparticles comprising sterol and acomponent derived from Quillaja saponaria Molina selected from quillajaacid and quillaja saponin, which nanoparticles do not comprise aphospholipid as an essential component. It also relates to a compositioncomprising the nanoparticles, and the use thereof, as adjuvant,especially in vaccines and as agents for treatment of cancer, ascarriers for amphipathic or hydrophobic molecules in the medical fieldespecially for treatment of cancer and for food related compounds.Further, it regards a method for producing the phospholipid-freenanoparticles, a method for the treatment of cancer and a method forassessing the applicability of the cancer treating method and for makingfood related compounds soluble in water to promote their uptake by thebody.

PRIOR ART

The Immune Stimulating Complex (ISCOM) is a 40 nm particle composed bysaponin from the tree Quillaja saponaria Molina that firmly associateswith cholesterol to form hexagonal rings with 6 nm diameter. The thirdcomponent is a lipid e.g. phosphatidyle choline that glues the rings toform a 40 nm spheres. This particle is used with the specific vaccineantigens incorporated into the particle or as an adjuvant particlewithout an antigen co-administered with the vaccine antigen in aseparate particle. The ISCOM particles may be produced with the methoddescribed by Lövgren & Morein and in EP 0 436 620 as well as inWO2004/004762.

One problem with the ISCOM and ISCOM Matrix is their complex productiontechnology. That also raises problems to use it as a carrier/deliverysystem e.g. to integrate molecules/compounds to be passenger or toachieve complimentary effects for pharmacological and vaccine effects oras a targeting device.

Vaccines are mostly based on whole microorganisms or subunits thatpromote immune responses, including both antibody and T cell responsesagainst surface structures. Alternatively, the vaccine antigens aresubunits, i.e. most often the surface proteins, but alsointernal/intracellular proteins or even non-structural proteins beingexpressed in cellular vectors. Surface proteins and carbohydrateantigens are often valued for their capacity to evoke antibodyresponses, not excluding that they also induce cell mediated includingT-cell responses, however, mostly not cytotoxic T-cell responses.Internal and non-structural proteins are used as vaccine antigens toevoke T cell responses including cytotoxic T-cells, since antibodies donot interact with internal proteins of the infecting agents and can,therefore, not mediate immune protection at the time point of infection.In contrast cell mediated immunity including T-helper cells andcytotoxic T-cells can kill infected cells i.e. after the time point ofinfection. Formulations and products of the ISCOM technology are used toenhance the immunogenicity of the accessible antigens i.e. surfaceantigens and the antigens revealed by the disruption (internal antigens)of the agent from which and against which the vaccine is prepared, cfMorein et al 2007³ and WO2011/005183. Any vaccine antigen can also beproduced by rDNA techniques and in many cases also syntheticallyproduced as described by Lövgren & Morein². The ISCOM technology isdescribed in a number of patent applications, including US 2006/0121065EP1539231A1, WO 2004/004762 and WO2005/002620).

Adjuvants in general are used to enhance level and quality of the immuneresponse of the antigens included in the vaccine formulation. However,there are a number of infections agents that an unmet need is prevailingregarding protective vaccines and that (new) that immune protection isescaped by:

-   -   Escape mutants (human influenza virus, corona virus in chicken        (infections bronchitis virus [IBR]), hepatitus C virus (HCV)    -   Not revealing antigenic determinants        -   Inaccessible-hidden (staphylococcus aureus [SA],            streptococcus equi)        -   Immune dominance by other antigenic determinants in the            microorganism exemplified by influenza virus in man,            parvovirus causing alution disease in mink, hepatitis C            virus (HCV) in man.        -   Inducing immune responses that exacerbate disease            (parvovirus [alution disease] mink)        -   Vaccines intended for species of pathogens having many to            almost innumerable variants making it difficult/too costly            alternatively making it more economical to produce a            sufficiently covering vaccine e.g. HIV and Hepatitis C in            man. It is also well-known that there are a number of            vaccines that need several even as many as up to 23 vaccine            components from the same number of strain variants e.g.            carbohydrate variants (conjugate vaccines e.g. Haemophilus            influenzae, Meningococus miningitides, Streptococcus            pneumonia, Streptococcus pyogenes, Pneumococcus pneumonie            and also Staphylococcus aureus) having various capsule            antigens.        -   Other unmet needs prevails for various Gramm+ cocci e.g.            Staphylococcus spp in animals particularly a need is            required for vaccines protecting against mastitis in            ruminants, caused by against S aureus, Streptococcus spp in            horse (Str. Equi, Str. Zooepidemicus), in cattle (Str.            agalacti, Str. dysgalacti and Str. Uberis)        -   Antigenic sin (FLU) or carrier induced epitope suppression            antigen (CIES).        -   Fast replicating agents e.g. Human immune deficiency virus            (HIV) complicates the escaping immune protective mechanisms            of the host by new upcoming variants including those induced            by vaccines.        -   DNA and RNA vaccines lack in many cases adjuvants

Thus, there is an unmet need to increase the capacity of vaccines tomeet upcoming situations that e.g. lead to epidemics and even more topandemics or to improve the possibility to keep protective value of avaccine by evading negative effects of escape mutation or to compensateimmunity lost by escape by mutation, or in enhance immune protection toupcoming variants due to mutations during the infection. For that reasonalso new particulate vaccine adjuvants may be required with theflexibility to adapt its steering of the immune response to animmunological profile required for protection against a particularpathogen.

Cancer is treated in various ways including surgery, irradiation and bypharmaceuticals, i.e. cytostatic drugs. The medical treatment generallyby cytostatic drugs cause severe side effects like irradiation therapyoften causing severe side effects. Thus, there is an unmet need to havea medical treatment that is well tolerated by the patients.International patent publication WO2008/063129 and Hu et al¹, describesnanoparticles comprising cholesterol, phosphatidylcholine and Quillajasaponin fractions ASAP (acyl-saponin, corresponding to QS 21 or QHC) orDSAP (desacyl-saponin, corresponding to QS 7 or QHA). These particlesnamed KGI and BBE are described to kill cancer cells at 30 to 40 foldlower concentrations than they are killing normal cells of similarorigin as described in the invention “Killcan, New Use” in the patientapplication PCT/SE 2007/050878 and WO2008/063129 and Hu et al¹. Theseparticles have similar production complexity as those described forISCOM and ISCOM Matrix.

Many potential pharmaceuticals cannot be developed became theresolubility in water could not be achieved including their use in thefields of vaccine/adjuvant and drug delivery including anticancerpharmaceuticals. If such potential pharmaceuticals were taken from shelfand rendered soluble in water some of those would enrich the medicalmarket.

SUMMARY OF THE INVENTION

The present inventors have identified a need for a new form ofnanoparticle to be used as anticancer pharmaceutical, carrier/deliveryparticle for pharmaceuticals and as adjuvant that can compensate for theshortcoming of commercially available adjuvant-vaccine formulations.

A problem with the ISCOM technology is the complex procedure toformulate the particles based on detergent solubilisation of theQuillaja components, cholesterol the third components e.g. phosphatidylcholine e.g. using ultra filtration, tangential flew, dialysis orcentrifugation techniques. All of those techniques as described byLövgren & Morein², cause loss of material during the production process.

Moreover, the ISCOM technologies are not readily suitable forintegration of other hydrophobic or amphipathic molecules since methodsso far developed allow the strong tendency of such compounds tospontaneously form stable complexes (self assembly) in water e.g.micelles not being integrated info the ISCOM formulation e.g. byhydrophobic interaction.

The present invention relates to a phospholipid nanoparticle comprisingsterol and at least one saponin.

In contrast to the present invention, lipid containing particles such asliposomes, ISCOM, ISCOM MATRIX, posintros and various kinds of liposomesfor the preparation and use in pharmaceuticals including adjuvantformulations to enhance the efficacy of vaccines and in vaccineformulations and formulations for treatment of cancer contains lipidslike phospholipids e.g. phosphatidylserin and phosphatidylcholine,stearylamin etc.

The nanoparticles according to the invention may also be used asdelivery systems for one or several compounds e.g. for pharmaceuticalsincluding those used for treatment of cancer and nutrition relatedcompounds where the additional substance(s) provide additional functionsand complementary modes of action.

The advantage of the nanoparticles according to the invention meritsthem as replacements for the prior art formulations including abroadened application as adjuvants to cover new variants of a pathogene.g. upcoming pandemics described above.

The present invention provides an easy production process with virtuallyno losses, due to the evaporation technology. That does not exclude theuse of techniques as described for the production of ISCOMs or ISCOMMatrix (see above).

Aspects of the invention are described in the independent claims.Preferred embodiments are set forth in the dependent claims.

SHORT DESCRIPTION OF THE APPENDING DRAWINGS

FIG. 1A. The electron microscopy (EM) shows a nanoparticle comprisingcholesterol, QHC and diterpenoid in a molar ratio of 1:1:0.5. Theparticles have a mean diameter of about 17-20 nm according to theinvention. It is distinctly different from an ISCOM particle of about 40nm as depicted FIG. 1B. Particles according to the invention without thediterpenoid have the same morphology.

FIG. 1B. The electron microscopy shows an ISCOM like particle comprisingcholesterol, QHC and phosphatidylcholin in a molar ratio: 1:1:0.5. Theparticles is prepared as described using a technology similar to thatdescribed in Example 1 according design C in Example 1 have a diameterof about 40 nm i.e. using the. The morphology and size are distinctlydifferent from those of a nanoparticle according to the invention asdepicted in FIG. 1A.

FIG. 2. Comparison of cancer cell killing capacity between G3 and KGInanoparticles both containing QHC tested on U937 cells. G3 and KGI havevirtually the same anti-cancer cell killing effect on the model tumourcell (P=0.8422).

FIG. 3. Comparison of G3 with KGI to inducing U937 tumour cells toproduce IL-8 showing that both particles have similar capacity to inducecancer cells to produce cytokine indicating cancer cell differentiation(P>0.05).

FIG. 4A. Stevia is the strongest inducer of IL-12B, IL-6 and IL-1alphacompared to BBE or KOI.

FIG. 4B. DT, when incorporated into G3, up-regulates cytokine IL-12 geneexpression of normal human DCs.

FIG. 5. G3 particles influence intra-cellular TK production of U937cells at a similar magnitude to that of KGI particles (the bars in eachformulation-concentration combustion indicates time points of 24, 48,72, 96 and 120 hours from the left to the right).

FIG. 6. The titration curves of G3, G3 with DT incorporated (G3-DT) andnon-particulate QHC read on HL-60 AML cells. The G3 and G3-DTformulations kill the AML cells more efficiently than freenon-particulate QHC.

FIG. 7. The stand alone and combination effects of G3 and cytarabine onHL-60 AML cells. G3 enhances ths significantly the cytarabine.

FIG. 8. G3 enhances the killing capacity of daunorubicin on HL-60 AMLcancer cells

FIG. 9. The titration curves of G3, G3 with DT incorporated (G3-DT) andQHC on PC-3 prostate cancer cells

FIG. 10. The stand alone and combination effects of G3 and docetaxel onPC-3 prostate cancer cells. The G3 enhances significantly the cancercell killing effect of docetaxel.

FIG. 11. G3 enhances the killing effect of cabazitaxel on PC-3 prostatecancer cells

FIG. 12. The titration curves on ACHN kidney cancer cells show that G3formulated with QHA is more potent (P<0.01) than G3 formulated with QHAin killing the solid cancer cells.

FIG. 13A. Immunization schedule

Experimental design (C56Bl6 mice, 6 mice/group, 200 μl/dose, s.c. twoimmunizations, 4 weeks apart)

Group 1 (Abisco Control): Influenza 1 μg+ISCOM−5 μg

Group 2 (G3-High Dose): Influenza 1 μg+G3−5 μg

Group 3 (G3-Medium Dose): Influenza 1 μg+G3−2.5 μg

Group 4 (G3-Low Dose): Influenza 1 μg+G3−1 μg

Group 5 (G3 with DT): Influenza 1 μg+G3−2.5 μg

Group 6 (Non-adjuvanted, Antigen Control): Influenza 1 μg

Group 7 (Non-immunized Control)

Evaluation

Blood for antibody responses. Spleen cells at necropsy for cell-mediatedimmunity including proliferation test, IL-4, IFN-γ.

FIG. 13B. HI antibody response of mouse serum measured at 3 weeks afterthe 1^(st) immunization

FIG. 13C. HI antibody response of mouse serum measured at 4 weeks afterthe 2^(nd) immunization

FIG. 13D. Cytokine responses of mouse spleen cells at 4 weeks after the2^(nd) immunization

FIG. 14A. The G3/VLX40 formulation (the right column) has high cancercell (U937) killing effect. In contrast to VLX40 alone (the left column)had low cancer cell killing effect indicating high solubility G3/VLX40formulation and low cancer cell killing effect of the VLX40-DMSOformulation

FIG. 14B. VLX40 DMSO formulation (left column) has high anticanceractivity in the precipitates. In contrast the scanty precipitate of theG3-VLX40 (right column) formulation had low anticancer cell activityindicating that the anticancer cell activity essentially was located tothe water phase.

DEFINITIONS

All terms and words in the present specification shall be construed ashaving the meaning usually given to them in the relevant art unlessspecifically indicated otherwise. For the sake of clarity, a few termsare defined below.

A vaccine formulation is a pharmaceutical formulation that is usedprophylactically and improves/enhances protective immunity to/againstone or more particular diseases. A therapeutic vaccine according to theinvention can be used to cure disease when an antigen specific for acomponent connected to the disease is included in the formulation withthe invention or, as is particular for cancer treatment, the antigen ispresent in the cancer/tumor. A vaccine includes an “antigen” thatelicits an immune response in the treated subject and, optionally, asubstance added to a vaccine to improve the immune response called an“adjuvant”.

An “antigen” is thus the active specific part in a vaccine, and may bethe entire micro-organism, such as virus or bacteria, causing thedisease that the vaccine is aimed at improving immunity to. It may alsobe a part of said micro-organism a subunit, such as a protein (asub-unit) a part of a protein, a protein either isolated from thepathogenic microorganism or produced by rDNA technique or syntheticallyproduced then often called peptide. A peptide has fewer amino acids thana protein.

An “adjuvant” is a vaccine constituent that enhances the level and orthe quality of the immune response to the antigen part of theprophylactic or therapeutic vaccine. A nutrition related compound is anycompound related to nutrient including vitamins health active substancestaste improving compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nanoparticles comprising sterol and acomponent from Quillaja saponaria Molina selected from quillaja acid andquillaja saponin, characterized in that said nanoparticles do notcomprise a phospholipid.

The resulting particle according to the innovation differs from ISCOMswith regard to size, wherein the particles according to the inventionare below 40 nm, around ˜20 nm whereas ISCOMs are ˜40 nm. Thus, the nanoparticle of the invention may have a diameter in the range of 10-40nanometers, preferably 12-35 nanometers or 15-25 nanometers. The sizewill to an extent dependent on the load of integrated other moleculesthan cholesterol and the quillaja molecule

The sterol may be selected from cholesterol, cholestanol, caprostanol,phytosterols, e.g. stigmasteroll sitosterol, mycosterols, e.g.ergosterol, preferably cholesterol and vitamin D3 or any hydrophobiccompound the is exposed in water to react covalently with reactive groupin the water soluble including suspension in water of a micelle.

Quillaja saponin or any of its fractions with a common triterpenoidskeleton also named Quillaja acid may be used. There are so far 4 formsor quillaja acids described. Quillaja saponins are forming chains with anumber of sugars either with an acyl group i.e. an acyl-saponin (ASAP)or without the acyl chain i.e. desacyl-saponin (DSAP) as described ine.g. Hu et al.¹

The saponin may be hydrophilic and selected from factions 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 and 15 of Quil A, especially fractions 7, 8, 9,10, 11, 12, 13 and 14 described in EP 0 3632 279 B2 and fraction A ofQuil A or crude Quil A.

The saponine may be a crude or raw, or non-fractionated extract of QuilA comprising a mixture of saponins or a semi purified forms thereof suchas Quillaja Powder Extract (Berghausen, USA), Quillaja Ultra Powder QPUF 300, Quillaja Ultra Powder QP UF 1000 or Vax-Sap (all three fromNatural Responses, Chile) or from Prodalysa, Santiago, Chile. Thepurified saponin fractions C and B solitary or combined together withmay be used. The B and C fractions are described in WO 96/11711, the B3,B4 and B4b fractions described in EP 0 436 620. The fractions QA1-22described in EP 0 3632 279 B2, Q-VAC (Nor-Feed, AS Denmark), QuillajaSaponaria Molina Spikoside (Isconova AB, Uppsala Science Park, 751 83,Uppsala, Sweden).

The saponin may be hydrophobic saponin and selected from saponins thatdo contain fatty acids e.g. in the 4-position in the triterpenoidaglycone of the saponins from Quillaja Saponaria Molina such as fractionC and B of Quil A or fractions from the region between fractions A and Band fractious 15, 16, 17, 18, 19, 10 and 21 described in EP 0 3632 279B2, especially fractions 17 and 18 are suitable here. Preferablyquillaja saponin fraction QHA, QHB and/or QHC may be used.

The ratio between cholesterol and quillaja saponin may be from 1:10 to10:1, preferably from 1:2 to 2:1.

The nanoparticles according the invention may further comprise at leastone an amphipathic or a hydrophobic molecule, which may be selected froman antigen, an adjuvant, a targeting molecule, a pharmaceutical compoundand a food related compounds.

The antigen may be any antigen with amphipathic or a hydrophobic groupsas described in EPC-patent application 0 109 942, or rendered to havehydrophobic region by rDNA expression and produced by cells orchemically synthesized. The adjuvant may be any adjuvant withamphipathic or hydrophobic groups such as those obtained from Quillajasaponaria Molina.

One or more compounds molecules may be incorporated into G3 forcomplementary functions e.g. as targeting device or as antigen orcomplementary antigens in the use of vaccines for immune modulatoryfunctions described in EP 9600647-3(PCT/SE97/00289 or as pharmaceuticalincluding anticancer or nutritional effects. To be incorporated into G3particles the molecules require hydrophobic domains or are electrostaticattached to the G3 particles. Compounds that do not have hydrophobicportions may be coupled to molecules having such molecules before orafter incorporation into the G3 particle as described for a similarparticle in EP 1800564.

Any adjuvant may be incorporated such as, natural or synthetic includingsynthetic or semi synthetic quillaja saponin or saponin fractions orderivatives thereof from Quillaja saponaria Molina, lipid A orderivatives or synthetic versions thereof, cell wall skeleton but notlimited to mentioned adjuvant compounds. A Diterpenoid (DT) supplied byJavier Saints, Prodalysa, Santiago, Chile may be used as an adjuvant anda nutritional (from stevia a sweetening agent). The diterpenoid (DT) hasbeen integrated into the nanoparticles according to the inventionresulting in typical small nanoparticles of 17 nm.

Lipid-containing receptors that bind to cell-binding components,including cholera toxin's receptor, which is the ganglioside GM1, andfocused blood group antigen may be used. The cell-binding components canalso function as mucus targeting molecule. The technology for complexescomprising are described in e.g. WO97/30728 and can be applied to G3particle both for anticancer treatment and for vaccine use. Anysubfragment of Quillaja saponaria Molina may be used solitary or invarious combinations. Receptors supplied with hydrophobic tail/regionintended to capturing molecules to the invented particle to supplydesired complementary properties e.g. different mode of cancer cellkilling e.g. monoclonal antibodies that both target cancer cells andalso have cell killing effect is one carrier-delivery option.

Thus other components that may be integrated into the nanoparticles arepharmaceuticals including anticancer drugs including receptors forantibodies or monoclonal antibodies such as Fc receptors or the DD ofProtein A of Staphylococcus aureus (WO2011/005183).

The production method of nanoparticles disclosed by the presentinvention is simpler than for ISCOM and more suited to incorporatehydrophobic and amphipathic molecules. Thus, the inventive nanoparticleis a nanoparticle suited for delivery of vaccine antigens, drugs foranticancer treatment as well as for any kind of drug. The particleproduced as described herein can also be supplemented with integratedamphipathic molecules (lipids such as stearylamine etc.) to be used forcovalent linking other molecules e.g. drugs or vaccine antigens, or forelectrostatic linking, lectin linking as described Morein etc³, and inWO2004/004762.

The particle may further comprise cancer targeting molecules such assurface antigens from cancer cells, virus surface antigens and influenzaantigens.

Surface molecules from microbial membranes may be incorporated byhydrophobic interaction as originally described by Morein et al³ and inEP 242380. Other molecules e.g. produced by rDNA technology orsynthetically produced can be incorporated as described in WO2002/080981 and WO 2004/030696.

Such targeting molecules include envelop proteins from viruses such asinfluenza and respiratory syncytial viruses having affinity torespiratory tract e.g. to target forms of lung cancer, or CTA1DD beingthe A1 part of the A subunit of cholera toxin incorporated into KGI orBBE formulations as described by Lycke et al⁴. CTA1DD is rationallydesigned of three main components, each contributing complementaryeffects. CTA1 is the enzymatically active subunit of cholera toxin thatis converted non-toxic by separation from the A2 and B subunits. Fusedto DD from protein A from Staphylococcus aureus it targets B cells. Moregenerally, mono and polyclonal antibodies can be incorporated into theparticles as described in EP 0 109 942 B1, EP 0 242 380 B1 and EP 0 180564 B1.

The invention also regards a composition comprising one or morenanoparticles. The composition may comprise different quillaja saponinfractions each incorporated in different nanoparticles.

Thus, two different saponin fractions may be complex bound in one G3particle and the other one (the other ones) of the at least twodifferent saponin fractions is (are) complex bound in another (other)physical different lipid containing particle(s).

The different saponins may be hydrophilic and hydrophobic saponinsrespectively. The particle may contain at least fraction C or at leastfraction B or at least any fraction between fraction C and B of Quil Aand at least one other fraction of Quil A. Thus one particle maycomprise fraction C only; fraction C and at least one other fraction ofQuil A; fraction C and one or more fractions of Quil A; fraction C andfraction A of Quil A; crude Quil A. The particle may also containfraction B only; fraction B and at least one other fraction of Quil A;fraction B and one or more fractions of Quil A; fraction B and fractionA of Quil A. The above combinations of fractions may also be indifferent lipid particle or in the same lipid particle.

Thus, mixtures of lipid containing particles comprising hydrophilic andhydrophobic saponins in physically different particles may be used.

According to one embodiment the fraction A of Quil A may be integratedinto a nano particle together with at least one other adjuvant withimmunomodulating activity.

According another embodiment the at least one other adjuvant is presentin free form or integrated into another separate nano particle for thepreparation of an adjuvant composition.

The at least one other adjuvant may be a saponin such as a Quil asaponin.

Fraction A may facilitates the use of another adjuvant which when usedby itself might be toxic in doses it is efficient and a synergisticeffect including enhancement of immune responses and immunomodulatingactivity may be obtained.

A composition according to the invention may comprise the adjuvantfraction A from Quil A and the at least one other adjuvant in any weightratios. Preferably fraction A of Quil A is from 2-99.9 weight %,preferably 5-90 weight % and especially 50-90 weight % counted on thetotal amount of adjuvants. For e.g. Al(OH)₃, oil adjuvants and blockpolymers the amount of fraction A, of Quil A may be substantially lower.

One preferred iscom composition comprises 50-99.9% of fragment A of QuilA and 0.1-50% of fragment C and/or fraction B and/or other fractions orderivatives of Quil A (hereinafter non-A Quil A fractions) counted onthe total weight of fractions A and non-A Quil A fractions. Especiallythe composition comprises 70-99.9% of fragment A of Quil A and 0.1-30%of non-A Quil A fractions, preferably 75-99.9% of fragment A of Quil Aand 0.1-25% of non-A Quil A fractions and especially 80-99.9% offragment A of Quil A and 0.1-20% of non-A Quil A fractions counted onthe total weight of fraction A and non-A Quil A fractions. Mostpreferred composition comprises 91-99.1% of fragment A of Quil A and0.1-9% of non-A Quil A fractions counted on the total weight offractions A and non-A Quil A fractions, especially 98.0-99.9% offraction A and 0.1-2.0% of non-A Quil A fractions counted on the totalweight of fractions A and non-A Quil A fractions.

The nanoparticles and a composition comprising the nanoparticles may beused as a pharmaceutical optionally in a pharmaceutical compositionfurther comprising pharmaceutically acceptable buffers, diluentsexcipients, additives, adjuvants and/or carriers.

Suitable pharmaceutically acceptable carriers and/or diluents includeany and all conventional solvents, dispersion media, fillers, solidcarriers, aqueous solutions, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art, and it is described, by way of example, in Remington'sPharmaceutical Sciences, 18th Edition, Mack Publishing Company,Pennsylvania, and USA. Except insofar as any conventional media or agentis incompatible with the active ingredient, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active ingredients can also be incorporated into thecompositions.

The invention also comprises a pharmaceutical composition furthercomprising at least one pharmaceutically active compound, such asanticancer drugs, platinum coordination compounds, taxane compounds,camptothecin compounds, anti-tumour vinca alkaloids, anti-tumournucleoside derivatives, nitrogen mustard or nitrosourea alkylatingagents, anti-tumour anthracycline derivatives, trastzumab andanti-tumour podophyllotoxin derivatives, Quillaja saponaria Molina andsub fragments thereof, receptors for antibodies or monoclonal antibodiessuch as Fe receptors or the DD of Protein A of Staphylococcus aureus,agents for treating cancer, such as agents selected from the groupconsisting of Cytarabin, Daunorubicin, Paclitaxel, Docetaxel,Cabazitaxel, Toricsel and Trabectidin, which active compound may beintegrated into the nanoparticle or mixed with the composition.

The further anti-cancer agents are preferably selected from namelyplatinum coordination compounds, taxane compounds, camptothecincompounds, anti-tumour vinca alkaloids, anti-tumour nucleosidederivatives, nitrogen mustard or nitrosourea alkylating agents,anti-tumour anthracycline derivatives, trastzumab and anti-tumourpodophyllotoxin derivatives.

The term “platinum coordination compound” is used herein to denote anytumour cell growth inhibiting platinum coordination compound whichprovides platinum in the form of an ion. Preferred platinum coordinationcompounds include cisplatin, carboplatin, chloro(diethlenetriamine-platinum (II) chloride; dichloro(ethylenediamine)-platinum (II); diamine(1,1-cyclobutanedicarboxylato)-platinum (II) (carboplatin); spiroplatin;iproplatin; diamine (2-ethylmalonato)-platinum (II); (1,2-diaminocyclohexane) malonatoplatinum (II); (4-carboxyphthalo) (1,2-diaminocyclohexane) platinum (II); (1,2-diaminocyclohexane)-(isocitrato) platinum (II); (1,2-diaminocyclohexane)-oxalate-platinum (II); ormaplatin and tetraplatin.

Cisplatin is commercially available for example under the trade namePlatinol from Bristol Myers Squibb Corporation as a powder forconstitution with water, sterile saline or other suitable vehicle. Otherplatinum coordination compounds and their pharmaceutical compositionsare commercially available and/or can be prepared by conventionaltechniques.

The taxane compound may be those sold under the trade name Taxol fromBristol Myers Squibb and docetaxel is available commercially under thetrade name Taxotere from Rhone-Poulenc Rorer. Both compounds and othertaxane compounds may be prepared in conventional manner for example asdescribed in EP 253738, EP 253739 and WO 92/09589 or by processesanalogous thereto. Carbazitaxel available from Sanofi Pasteur.

Camptothecin compounds include irinotecan and topotecan. Irinotecan iscommercially available for example from Rhone-Poulenc Rorer under thetrade name Campto and may be prepared for example as described inEuropean patent specification No. 137145 or by processes analogousthereto. Topotecan is commercially available for example from SmithKlineBeecham under the trade name Hycarntin and may be prepared for exampleas described in European patent specification No. 3121122 or byprocesses analogous thereto. Other camptothecin compounds may beprepared in conventional manner for example by processes analogous tothose described above for irinotecan and topotecan.

Anti-tumour vinca alkaloids include vinblastine, vincristine andvinorelbine referred to above. Vinblastine is commercially available forexample as the sulphate salt for injection from Eli Lilly and Co underthe trade name Velban, and may be prepared for example as described inGerman patent specification No. 2124023 or by processes analogousthereto. Vincristine is commercially available for example as thesulphate salt for injection from Eli Lilly and Co under the trade nameOncovin and may be prepared for example as described in the above Germanpatent specification No. 2124023 or by processes analogous thereto.Vinorelbine is commercially available for example as the tartrate saltfor injection from Glaxo Wellcome under the trade name Navelbine and maybe prepared for example as described in U.S. Pat. No. 4,307,100, or byprocesses analogous thereto. Other anti-tumour vinca alkaloids may beprepared in conventional manner for example by processes analogous tothose described above for vinoblastine, vincristine and vinorelbine.

Anti-tumour nucleoside derivatives include 5-fluorouracil, gemcitabineand capecitabine referred to above. 5-Fluorouracil is widely availablecommercially, and may be prepared for example as described in U.S. Pat.No. 2,802,005. Gemcitabine is commercially available for example fromEli Lilly under the trade name Gemzar and may be prepared for example asdescribed in European patent specification No. 122707 or by processesanalogous thereto.

Capecitabine is commercially available for example from Hoffman-La Rocheunder the trade name Xeloda and may be prepared for example as describedin European patent specification No. 698611 or by processes analogousthereto. Other anti-tumour nucleoside derivatives may be prepared inconventional manner for example by processes analogous to thosedescribed above for capecitabine and gemcitabine.

Nitrogen mustard compounds include cyclophosphamide and chlorambucil.Cyclophosphamide is commercially available for example fromBristol-Myers Squibb under the trade name Cytoxan and may be preparedfor example as described in U.K. patent specification No. 1235022 or byprocesses analogous thereto. Chlorambucil is commercially available forexample from Glaxo Welcome under the trade name Leukeran and may beprepared for example as described in U.S. Pat. No. 3,046,301, or byprocesses analogous thereto. Preferred nitrosourea compounds for use inaccordance with the invention include carmustine and lomustine referredto above. Carmustine is commercially available for example fromBristol-Myers Squibb under the trade name BiCNU and may be prepared forexample as described in European patent specification No. 902015, or byprocesses analogous thereto. Lomustine is commercially available forexample from Bristol-Myers Squibb under the trade name CeeNU and may beprepared for example as described in U.S. Pat. No. 4,377,687, or byprocesses analogous thereto.

Anti-tumour anthracycline derivatives include daunorubicin, doxorubicinand idarubicin referred to above. Daunorubicin is commercially availablefor example as the hydrochloride salt from Bedford Laboratories underthe trade name Cerubidine, and may be prepared for example as describedin U.S. Pat. No. 4,020,270, or by processes analogous thereto.

Doxorubicin is commercially available for example as the hydrochloridesalt from Astra, and may be prepared for example as described in U.S.Pat. No. 3,803,124 or by processes analogous thereto. Idarubicin iscommercially available for example as the hydrochloride salt fromPharmacia & Upjohn under the trade name Idamycin, and may be preparedfor example as described in U.S. Pat. No. 4,046,878 or by processesanalogous thereto. Other anti-tumour anthracycline derivatives may beprepared in conventional manner for example by processes analogous tothose described above for daunorubicin, doxorubicin and idarubicin.

Trastzumab is commercially available from Genentech under the trade nameHerceptin and may be obtained as described in U.S. Pat. No. 5,821,337 orPCT patent specifications WO 94/04679 and WO 92/22653.

Anti-tumour anti-tumour podophyllotoxin derivatives include etoposideand teniposide. Etoposide is commercially available for example fromBristol-Myers Squibb under the trade name VePesid, and may be preparedfor example as described in European patent specification No. 111058, orby processes analogous thereto. Teniposide is commercially available forexample from Bristol-Myers Squibb under the trade name Vumon and may beprepared for example as described in PCT patent specification No. WO93/02094, or by processes analogous thereto. Other anti-tumourpodophyllotoxin derivatives may be prepared in conventional manner forexample by processes analogous to those described above for etoposideand teniposide.

Saponins in crude form or fractions thereof such as those mentionedabove may also be used in free form, i.e. not integrated into lipidcomprising particles, as anti-cancerous agents. These anticancercompounds may be with, coupled on to or integrated into the lipidcontaining particles such as liposomes, iscom and/or iscom matrix andposintros.

It is suitable if they are hydrophobic when integrated. If nothydrophobic groups may be coupled on to them as described in EP 242380.

Non-hydrophobic compounds and especially proteins or peptides may berendered hydrophobic by coupling hydrophobic groups to them.

The hydrophobic group that can be coupled to the non-hydrophobiccompounds are straight, branched, saturated or unsaturated aliphaticchains, preferably having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbonatoms, or hydrophobic amino acids or peptides or other hydrophobicstructures such as steroids. The length of the hydrophobic structure isadapted to the size and nature of the protein. As an example, it can bementioned that a peptide with 10-15 amino acids (foot-and-mouth diseasevirus) suitably is brought out with two tyrosine at the amino or carboxyterminal end. A protein with a molecular weight of 70,000 daltonsdemands about 20 hydrophobic amino acids. Testing is made empirically.Thus, one uses especially peptides with 1 to 20 amino acids, preferably1, 2, 3, 4, 5 amino acids, especially chosen among Trp, Ile, Phe, Pro,Tyr, Leu, Val, especially Tyr; cholesterol derivatives such as cholineacid, ursodesoxycholine acid.

These hydrophobic groups must be bonded to a group that can be coupledto the non-hydrophobic protein or compounds such as carboxyl-, amino-,disulphide-, hydroxyl-, sulohydryl- and carbonyl group, such as aldehydegroups.

As hydrophobic groups that can be coupled are selected preferablycarboxyl, aldehyde, amino, hydroxyl, and disulphide derivatives ofmedian, ethane, propane, butane, hexane, heptane, octane, and peptidescontaining Cys, Asp, Glu, Lys, preferably octanal andTyr.Tyr.Tyr-Cys,-Asp or -Glu. The hydrophobic groups with a group thatcan be coupled must be dissolved in water with the aid of for examplethe solubilising agents and detergents mentioned above or hydrochloricacid acetic acid 67% by volume acetic acid, caustic liquor, ammonia,depending on what substance is to be dissolved. pH is then adjusted tothe neutral direction without the substance precipitating; here it is tomake sure that there is not obtained a pH value that denaturates theprotein to which the hydrophobic group is to be coupled. Lipid mayenhance the solubilisation.

The hydrophobic molecule may be added to the non-hydrophobic compound inthe molar ratio of 10:1 to 0.1:1, preferably 1:1.

Hydrophobic groups with a carboxyl group as coupling molecule can becoupled to the protein through water-soluble carbodiimides or compositeanhydrides. In the first case the carboxyl group is activated at pH 5with carbodiimide and mixed with the protein dissolved in buffer pH 8with a high phosphate content. In the latter case the carboxy compoundis reacted with isobutylchloroformate in the presence of triethylaminein dioxane or acetonitrile, and the resulting anhydride is added to theprotein at pH 8 to 9. It is also possible to convert the carboxyl groupwith hydrazine to hydrazide which together with aldehydes and ketones inperiodate-oxidized sugar units in the protein gives hydrazone bonds.

The amino groups with nitrous acid can at a low temperature be convertedto diazonium salts, which gives azo bonds with Tyr, His and Lys. Thehydroxyl groups with succinic anhydride can be converted tohemisuccinate derivatives which can be coupled as carboxyl groups.Aldehyde groups can be reacted with amino groups in the protein to aSchiff's base. Several coupling groups and methods aredescribed^(6,7,8).

The proteins, peptides or compounds so produced having receivedhydrophobic groups are then complex-bonded with glycoside, as describedin a) but here the purification steps for removing cell fragments can beomitted.

Hydrophilic proteins having enclosed hydrophobic groups can be renderedhydrophobic by making the hydrophobic groups accessible by gentlydenaturating the proteins, i.e. with a low pH of about 2.5, 3M urea orat a high temperature above 70.degree. C. Such proteins may beimmunoglobulines such as IgG, IgM, IgA, IgD and IgE. Theimmunoglobulines can be used as antidiotypic antibodies. The proteinsare obtained purified as proteins as described in (b) and thencomplex-bonded to glycoside as described in (a), the purification stepsfor removing cell fragments being omitted.

The hydrophobic or amphiphatic molecule may also be chosen fromphospholipides such as derivatives of glycerol phosphates such asderivatives of phosphatidic acids i.e. lecithin, cephalin, inositolphosphatides, spingosine derivatives with 14, 15, 16, 17, 18, 19 and 20carbon atoms, phosphatidylethanolamine, phophatidylserine, phosphatidylcholine.

All above mentioned amphipathic and hydrophobic molecule, which may beselected from an antigen, an adjuvant, a targeting molecule, apharmaceutical compound and a nutriment may be integrated into thenanoparticle or mixed therewith in a composition.

The pharmaceutical composition may be used as an adjuvant, e.g. for usein combination with a vaccine under development, for use in combinationwith a seasonal influenza virus vaccine, for use in combination with apandemic influenza vaccine or for use in combination with an emergencyvaccine, such as a vaccine against a biological weapon. Thus, theinvention also regards a pharmaceutical vaccine formulation comprisingthe G3 particles as mentioned above.

The invention also relates to a method for treating or preventing adisease caused or complicated by an organism, comprising administeringto a subject a pharmaceutical vaccine formulation according to theinvention to a person in need thereof.

Further, the invention regards a method for treatment of cancer,comprising administering to a patient in need thereof a pharmaceuticallyeffective amount of nanoparticles or a composition according to theinvention. According to one embodiment the said cancer is leukemia.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents, which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parentally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water.Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or deglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The solutions or suspensions could also comprise at least one of thefollowing adjuvants: sterile diluents such as water for injection,saline, fixed oils, polyethylene glycols, glycerol propylene glycol orother synthetic solvents, antibacterial agents such as benzyl alcohol ormethyl paraben antioxidants such as ascorbic acid or sodium bisulfite,chelating agents, such as ethylene diamine tetraacetic acid, bufferssuch as acetates, citrates or phosphates, and agents for adjustment ofthe tonicity such as sodium chloride or dextrose. The parenteralpreparation could be enclosed in ampoules, disposable syringes ormultiple dosage vessels made of glass or plastic.

The compounds of general formula may be administered parenterally. Theterm parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intradermal injection of infusiontechniques, electroporation (EP), for needle less injection—jetinjection, gene gun, biljector as well as oral, aerosol administrations.For oral use e.g. protein A derived compound CTA1DD may be used asdescribed by Eliasson etal.⁹ having a property to targeting B-cellsuseful treating-cells for induction of mucosal immunity particularly inthe intestinal tract but also poentially also for cancers particularlyfor B-cells lymphoma.

Generally, the lipid containing particles of this invention areadministered in a pharmaceutically effective amount. The amount of theparticles actually administered will be typically determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound administered, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

The nanoparticle according to the invention may be used as an adjuvantin any vaccine against any microorganisms. I may be used on any animalsuch as birds, mammals such as humans, domestic animals such as cats,dogs, sheep, goat, pigs, cattle and horses. According to one embodimentthe invention is used as adjuvant in a vaccine against Streptococci inanimals and influenza in horses.

Doses for human use may vary according to other compounds included. Inview of duration of treatment the dose may range from <50 μg to 1 mg ormore per day.

The invention also regards a method for assessing the applicability ofthe method for treatment of cancer according to the invention to anindividual patient, comprising:

-   -   bringing cancer cells from said patient in contact in vitro with        nanoparticles according to any one of claims 1-7 or a        pharmaceutical composition according to claim 8 or 9;    -   measuring at least one effect indicative or therapeutic effect        of said nanoparticles or pharmaceutical composition on said        cancer cells;        wherein the method according to claim 10 or 11 is assessed as        applicable to said individual patient if the nanoparticles or        pharmaceutical composition shows a significant effect indicative        of therapeutic effect on said cancer cells.

The indication of therapeutic effect can be read by down regulation ofgenes, which have importance in the cell cycle regulation as cyclinedependent kinases (CDKs), cyclins or other molecules promoting passageover check points in the cell cycle and replication (CDK2, CDK6 andCyclineD1) or down regulation of thymindine kinase (TK) and upregulationof molecules facilitating the cell differentiation, as IL-8, FOXC1 andHDAC5 also indicating exit from the cell cycle. The regulation factorsare examples and there are more i.e. the examples does not concludelimitations.

The invention also regards a method for producing phospholipid-freenanoparticles comprising the steps

-   -   a) providing a hydrophobic surface or a suspension of liposomes    -   b) bringing hydrophobic surface or the suspension of liposomes        into contact with a solution of sterol, preferably cholesterol        dissolved as monomers in an organic solvent or detergent;    -   c) removing the solvent or detergent forming a sterol membrane        on the surface    -   d) providing a water solution of quillaja saponin micelles    -   e) adding the water solution comprising the saponin micelles to        the sterol membrane, whereby a complex is formed between the        saponines and the sterols and is suspended in the water        solution.

The hydrophobic surface might be a surface in a jar, tub, several layersurfaces, beads, e.g. latex beads, nets or three dimensional, nets orporous material. It might also be a liposome with the componentsintegrated in the lipid membrane(s).

The liposomes can be constructed according various techniques and withdifferent compositions as described (e.g. in Review LiposomesPreparation Methods Mohammad Riaz Faculty of Pharmacy, University of thePunjab, Lahore, Pakistan Pakistan Journal of Pharmaceutical SciencesVol. 19(1), January 1996, pp. 65-77). It might also be constructed as avirosome containing virus proteins integrated in the liposome membrane.The liposomes may be in a water solution. The devices may e.g. be packedin columns.

The saponins and sterols may be the ones mentioned above for thenanoparticles

The solvent may be any solvent as may be found on the sitehttp://en.wikipedia.org/wiki/Organic solvents or detergent, preferablychloroform, ethanol or acetonitril. The nature of the solvent isdescribed in en.wikipedia.org/wiki/organic solvents. The selection ofsolvent is dependent on the nature of the molecule to dissolve. Thedifferent types of solvent are mainly classified according to polarityand non-polarity. Non-polar solvents are e.g. haxane, chlorform anddiethyl ether. Those mentioned are useful because the can be evaporatedhaving boiling points between 35 and 65 facilitating removal byevaporation. Polar aprotic solvents are often used for solubilization ofpharmaceutical molecules e.g. demethy sulfoxide, acetonitril. The lateris of interest because it has low boiling point. Polar protic solventare also useful particularly in combination with other solvents.Ethanol, methanol have low boiling point and acetic acid has highboiling point. Low boiling point is particularly important for theevaporation technique. The solubilisation may be done with two or moresolvents. The solvents mentioned are examples and there are many morehaving perhaps even more desired properties for the use in theinnovation for forming G3 formulations.

Examples of usable are as non-ionic, ionic i.e. cationic or anionic orZwitter-ionic detergent such as Zwittergent or detergent based on gallicacid which is used in excess. Typical examples of suitable non-ionicdetergents are N-alkanoyl-N-alkyl-glucamines, polyglycol esters andpolyglycol ethers with aliphatic or aralylphatic acids and alcohols.Examples of these are alkylpolyoxyethylene ethers with the generalformula C_(n)H_(2n+1)(OCH₂CH₂)_(x)OH, shortened to Cn Ex; alkyl-phenylpolyoxyethylene ethers containing a phenyl ring between the alkyl groupand the polyoxyethylene chain, abbreviated Cn phi.Ex, TritonX-100=tertC₈E₉6 (octylphenolether of polyethylene oxide),acylpolyoxyethylene esters; acylpolyoxyethylene sorbitane esters,abbreviated Cn sorbitane Ex, e.g. Tween 20, Tween 80,.beta.-D-alkylglucosides, e.g. .beta.-D-octylglucoside. Typical examplesof suitable ionic detergents are gallic acid detergents such as e.g.cholic acid, desoxycholate, cholate and CTAB (cetyltriammonium bromide).Even conjugated detergents such as e.g. taurodeoxyoholate,glycodeoxycholate and glycocholate can be used. Other possiblesolubilizing agents are lysolecithin and synthetic lysophosophoilipids.Even mixtures of the above-mentioned detergents can be used. When usingthe dialysis method the detergents should be dialysable in not too lingtime.

Some surface active substances greatly facilitate matrix formation.These include the intrinsic biological membrane lipids with a polar headgroup and a non-polar aliphatic chain e.g. phosphatidyl choline(negatively charged) and phosphatidyl ethanolamine (positively charged).

According to one embodiment the detergent may be Triton-X-100, Tween-20,Nonidet, NP-40, deoxycholate, MEGA-10 and octylglycoside. MEGA-10 andoctylglycoside can be removed by dialysis. For others other technologiescan be used as mentioned e.g. the centrifugation method and columnchromatography.

The soluble agent might be removed by evaporation using an organicsolvent with low boiling point or by dialysis or by dialysis,chromatography, filtration or tangential flow as described in EPC-patent0 109 942.

The water solution of saponin micelles is obtained by adding a freeze orspray dried powder as delivered from the producer. The saponin orsaponin fraction is normally kept as stock solution e.g. 10 mg/ml waterbut not limited to that concentration and added to the water surroundingthe lipid membranes at a final concentration above CMC i.e. criticalmicelle concentration e.g. 30 mg/liter exact figure is dependent on thequillaja product. The saponins are obtained as Quillaja Powder Extractand may be obtained from as crude quillaja extract (Berghausen, USA).Quillaja Ultra Powder QP UF 300, Quillaja Ultra Powder QP UF 1000 orVax-Sap a non-fractionated quillaja saponin product, QHA and QHCfractions (all three from Natural Responses, Chile) or from Prodalysa,Santiago, Chile.

The invention is using a new production method wherein an artificialmembrane of the sterol attached to a hydrophobic surface is produced insteps a-c). A wafer soluble micelle of a quillaja saponin product and achemical (covalent) binding between the quillaja micelle and a componentin the artificial membrane extracts the artificial membrane componentsinto a water soluble complex as an innovative water solublenanoparticulate complex i.e. G3. This complex is a held together by achemical (covalent) linking keeping and binding the hydrophilic partstogether in the water phase and hydrophobic interactions betweencomponents remains in the center of the complex. The membrane is formedfrom an organic solution with a soluble agent that may be a detergent oran organic solvent.

Hydrophobic and amphipathic molecules so be incorporated into theartificial membrane, are solubulised with organic solvent or detergenttogether with the sterol in step b) and transferred to waterphase byevaporation of the organic solvent. Dependent on the solvent it isremoved by evaporation if the boiling point is below that of water or bydialyses or by similar techniques described for ISCOM formation. Thus,the removal of the solvent is not a part of the formation of theparticle but for the formation of the artificial membrane that is not apart of the formation of the particles. Subsequent to the formation ofthe artificial membrane is completed there is water surrounding theartificial membrane. Into this water phase quillaja micelles suspended(dissolved) in water are added and the artificial membrane is extractedin the water phase and reorganization of the quillaja micelle to the newG3-formulation. The composition can readily be adjusted to completelydissolve the artificial membrane into a particulate suspension in water.The composition differs from a micelle from the construction point ofview that a covalent linking is involved thus an innovative particle isformed.

In steps f) and g) the water soluble micelle form of the quillajaproduct is allowed to interact to get the final product into a waterphase. The first interaction in this step is a covalent binding betweenthe quillaja micelle and the sterol in the artificial membrane and thesecond interaction is between the quillaja triterpenoid skeleton and thesterol. Under suitable proportions all compounds in the artificialmembrane are incorporated into water soluble quillaja micelle forming anew nanoparticle that will vary in size from 17 nm up to 40 nm. A largersize is obtained if lipids e.g. a phospholipid are present in theartificial membrane. The examples 2, 4, 9, 14, 15 and 16 demonstratethat various kinds of lipophilic molecules have been incorporatedaccording the invention including DT, busulfan, roscovitine, vivolux 40and vitamin D3.

Iscom matrix may be produced with the new method by adding at least onephospholipide to the suspension comprising sterol in step b).

The pbospholipide may be chosen from derivatives of glycerol phosphatessuch as derivatives of phosphatidic acids i.e. lecithin, cephalin,inositol phosphatides, spingosine derivatives with 14, 15, 16, 17, 18,19 and 20 carbon atoms, phosphatidylethanolamine, phophatidylserine,phosphatidyl choline.

Hydrophobic components may incorporated in step b) i.e. in theartificial membrane that includes also lipids, sterols.

In step d) the saponin in the water soluble form i.e. micelle form isadded to the water phase covering the artificial membrane.

The invented nanoparticle replaces the ISCOM matrix because it simplerand more economical to produce because the nanoparticle according to theinvention is based on two components i.e. a quillaja saponin,cholesterol in contrast to the ISCOM particle formation includes threecomponents i.e. a quillaja saponin, cholesterol and the third componenta phospholipid e.g. phosphatidyl choline. The quillaja component(s) neednot to be solubilized with detergent or with an organic solvent. The newproduction techniques according to the invention is robust and thesensitive balance is overcome. Thus, the new method is more suitablethan ISCOM matrix technologies for integration of a fourth, fifth ormore i.e. other hydrophobic or amphipathic molecules since methods sofar developed allow the strong tendency of such compounds tospontaneously form stable complexes (self assembly) in water e.g.micelles and therefore not being integrated into the ISCOM matrixformulation e.g. by hydrophobic interaction. Thus, the ISCOM matrixtechnology has shortcomings to be developed as a general deliverysystem, but the invention does not have such shortcomings.

All publication mentioned herein are hereby incorporated as reference.The invention will now be described by the following non-limitingexamples.

EXAMPLES Materials and Methods Chemical and Compounds

Cholesterol (C8667), phosphatidylcholine (PC, P-5763) and chloroform(288306) were all purchased form Sigma-Aldrich Sweden AB, Stockholm,Sweden, Fraction A (QHA) and Fraction C (QHC) of Quillaja saponin areall purchased from ISCONOVA AB, Uppsala, Sweden, Diterpenoid (DT) i.e.Stevia was obtained from Prodalysa Ltda., Chile, Vitamin D3 wascommercially obtained from Miva Nutri-molecular Research Limited,Shanghai, China.

Cell Lines

The human macrophage (Mφ) cell line U937 (which is often used as a modelcell line in biological and cancer research) and the human Acute MyeloidLeukemia (AML) cell lines HL-60 were grown in culture medium RPMI-1640.The human prostate adenocarcinoma, PC-3, cultured in a 50/50 mixture ofHAM's F-12K and RPMI-1640. All the cells were kindly supplied by thedivision of clinical pharmacology, Uppsala University. All media weresupplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mMglutamine, 100 μg/ml streptomycin and 100 IE/ml penicillin (all fromSigma Aldrich Co, St Louis, Mo., USA). All cell lines were incubated at37° C. in humidified air containing 5% CO₂.

Human Dendritic Cells (DCs)

Immature human DCs were purchased from 3H Biomedical, Uppsala, Sweden.

In Vitro Assay Procedure

Cells in 96-well micro-titer plates at a cell density of 5,000-20,000cells/well were exposed to serial diluted G3, KGI and Quillaja saponinproducts containing the same amounts of QHC at 37 C in humidifiedatmosphere containing 5% CO2 for 72 hours. For U937 cells, one set of,the cells were used directly for the fluorometric microculturecytotoxicity assay (FMCA) to measure cell killing effect of theformulations. For the other set of the cells, the supernatant wascollected 150 μl/well for cytokine IL-8 determination.

Measurement of Cancer Cell Killing Effect

The FMCA method is based on measurement of fluorescence generated fromhydrolysis of fluorescein diacetate (FDA) to fluorescein by cells withintact plasma membranes. After above mentioned incubation for 3 days,the medium was removed by aspiration. After one wash with PBS, 100μl/well of FDA dissolved in a physiological buffer (10 μg/ml) was added.The plates were incubated for 45 minutes and the generated fluorescencefrom each well was measured in a 96-well scanning fluorometer. Thefluorescence is proportional to the number of intact cells in the well.Quality criteria for a successful analysis included a fluorescencesignal in the control wells of more than five times of the mean blankvalue, a mean co-efficient of variation (CV) in the control wells ofless than 30%.

Cytokine IL-8 Determination for U937 Cells Stimulated with G3Formulations

ELISA for the detection of human IL-8 was carried out according to themanufacturer's instruction (Human IL-8 ELISA, catalog No. S8000C, R&Dsystem, Minneapolis, Minn. 55413, USA). Briefly, 50 μl reconstitutedstandards of human IL-8 and the supernatants were added to each well intriplicate wells and mixed well by gently tapping the plates severaltimes. The plates were then covered with adhesive plate covers andincubated for one hour at room temperature (RT, 20-25° C). After theincubation, the plates were washed 3 times with Wash Buffer and 50μl/well of the Biotinylated Antibody Reagent (anti-human IL-8) wasadded. The plates were covered again with adhesive plate covers andincubated for one hour at RT. After being washed 3 times with WashBuffer, 100 μl/well of Streptavidin-HRP Solution was applied. The plateswere covered with the adhesive plate covers again and incubated for 30minutes at RT. The contents in the plates were discarded and the plateswere washed 3 times with Wash Buffer. 100 μl of TMB Substrate Solutionwas dispended into each well. The enzymatic color reaction was allowedto develop at RT in the dark for 30 minutes. The reaction was stopped byadding 100 μl/well of Stop Solution. The absorbance was read on an ELISAplate reader at 450 nm and 550 nm. Subtract 550 nm from 450 nm values tocorrect for optical imperfections in the microplates. The standard curvewas then generated and used to calculate the amount of human IL-8 in theunknown samples. The standard curve was created by plotting the averageabsorbance obtained for each standard concentration on the vertical (Y)axis vs. the corresponding concentration (pg/ml) on the horizontal (X)axis.

Cytokine IL-12 Gene Expression of Human Monocytes Stimulated by G3Formulation

The cytokine IL-12 gene expression of treated DCs by G3, DT, G3 with DTincorporated were compared with the cell control by gene arrays.Briefly, normal human monocytes were exposed to 10 μg/ml of G3, 100μg/ml of DT and the combination of these two in the same particles withthe same concentrations for 6 hours, then RNA was isolated according tothe manufactures manual (QIAGEN RNeasy Minikit). RNA expression analysiswas done at “Uppsala Array Platform, Clinical Chemistry andPharmacology, Uppsala University Hospital Uppsala-Sweden” by convertingthe RNA samples to labeled cDNA via reverse transcription and comparingthe quantitative data from the various samples with untreated cells(Ambion WT Expression Kit).

Thymidine Kinase (TK) Activity

The TK activity was determined with a kit obtained from Biovica(Uppsala, Sweden). Briefly, after exposing to KGI or G3 formulations atvarious time points, 100 μl cell suspension at a concentration of0.1-1×10⁶ cells/ml was transferred to Eppendorf tubes and centrifuged at200 g for 10 minutes. The cell pellet was re-suspended in 100 μl coldPBS and freeze/thawed 2-3 times. After centrifugation at maximum speedfor five minutes, then the cells were collected. The inter-cellular TKactivity was measured according to the manufacturer's protocol.

Detection of Vitamin D3

Samples with cholecalciferol (vitamin D3) incorporated in G3 particleswere analysed at a University Hospital Laboratory on a Liaison automaticinstrument. Although the assay (DiaSorin Liaison) is designed to measure25-HO-D3 it has about one percent cross-reactivity with non-hydroxylatedvitamin D3.

Influenza Virus Strains and Vaccine

The human influenza virus A/California/07/2009 (H1N1), APerth/16/2009(H3N2) and B/Brisbane/60/2008(B) as a non-adjuvantedvaccine was used as antigen in the preparation of the vaccines, theserological tests and in the re-stimulation of lymphocytes. The viruswas cultured on VERO cells and split with deoxycolate. It was kindlysupplied by the manufacturer. After harvest, the viruses were purified,inactivated, split and re-suspended at a concentration of 30 μgprotein/ml. The dose contained 1 μg virus antigen and various amount ofadjuvant as indicated in FIG. 1.

Vaccination

C56B16 mice hosted at the animal facility, University Hospital,Karolinska Institute, Stockholm, were immunized subcutaneously in theneck twice. For details, see example 12.

Haemagglutination (HA) Test

Chicken erythrocytes (RBCs) collected in citrate solution were washed 3times using 0.01 M phosphate buffered saline (PBS) pH 7.2 andre-suspended at a concentration of 0.5% in PBS containing 0.05% bovineserum albumin (BSA). The HA test was performed in U-type microplates at4° C. for 1 hours.

Haemagglutination Inhibition (HI) Test

Serum samples were incubated at room temperature (RT°) together with a30% suspension of chicken RBCs for 1 hour (h). After absorption, themixtures were centrifuged at 500×g for 10 min and the supernatantscollected. The final serum dilution was 1:5. The HI test was carried outusing V-type microplates and 16 HA-units/50 μl. Serum samples, 25 μlwere 2 folds diluted using an equal amount of PBS-BSA. The diluted serawere incubated at RT° for 1 h together with 25 μl of virus suspensionafter which the mixtures were incubated at 4° C. for 1 h. The highestserum dilution inhibiting 100% the Haemagglutination was considered asthe antibody titer for the sample.

Preparation of Lymphocytes

The spleen-lymphocytes (splenocytes) were obtained as aseptically aspossible. Immunized and non-immunized mice were bled and sacrificed bycervical dislocation at 3 weeks post revaccination. Spleens were removedand thereafter carefully teased, passed through a sterile stainlesssteel mesh and flushed with EMEM with Tricine using a pipette. The cellswere washed twice using EMEM with Tricine means centrifugation at 500×gwith. Then, the pellets were re-suspended in F-DMEM medium supplementedwith 1% fetal calf serum (FCS), 10 μg gentamicin/ml, 2 mMl L-glutamine,3.81 g Hepes/L and 5×10⁻⁵ M β-mercaptoethanol (culture medium). Thecells viability was assayed by Trypan blue dye exclusion test.

Enzyme-Linked Immunespot Assay (ELISPOT)

The enumeration of cytokine secreting splenocytes was carried out usingcommercial ELISPOT-kits for INF-γ, IL-2 or IL-4. The kits were purchasedfrom Mabtech, Stockholm, Sweden. The ELISPOT plates were used followingthe instructions recommended by Mabtech.

For each cytokine, splenocytes at a concentration of 2×10⁵ per 100 μlculture medium were pipette into 8 different wells. Four replicatesreceived 50 μl culture medium containing 4.5 μg haemagglutinin ofinfluenza virus antigen. The resting four wells received 100 μl ofculture medium only. Plates were incubated at 37° C. in humidified boxesfor 18 h after which the cells were discarded and the wells washed.Spots were developed following the procedure described by Mabtech. Inshort, plates were incubated for 2 h as RT° with 100 μl biotinylatedmonoclonal antibodies (MoAb) anti IFN-γ, IL-2 or IL-4. Then, the plateswere carefully rinsed and thereafter incubated for 1 h at RT° with HRPOconjugated Streptavidin. After another wash cycle, the plates wereincubated with the substrate at RT° for approximately 15 min or untildistinct spot emerged. Washing the plates with tap water stopped thereactions. Finally, the plates were allowed to dry and thereafter thenumber of spots was counted using an ELISPOT counter.

Data Analysis and Statistics

Dose-response data were analyzed using calculated SI values and thesoftware program GraphPadPrism4 (GraphPad Software Inc. San Diego,Calif. USA). Data are presented as mean values±SE. Statisticalinferences between several means were performed by one-way ANOVA withTukey's multiple comparison post test of group means and for comparisonof two means, by Student's t-test, in GraphPadPrism.

Part I. Formulation and Characterization Example I

In this example, the formulation of the G3 nanoparticles is described.In experimental set up step 2, A and B, (see below) the influence of theproportions of cholesterol vs QHC fraction (from ISCONOVA AB, Uppsala,Sweden, see WO2008/063129) of Quillaja saponin has been explored. In Cthe effect of adding a phospholipid is explored with regard to particleformulation.

Experimental Set-Up

In step 1 an artificial cholesterol membrane is formed requiring asolubilisation in detergent or organic solvent. In this experiment wehave used chloroform (288306, Sigma-Aldrich Sweden AB, Stockholm,Sweden) as the solvent for cholesterol (C8667, Sigma-Aldrich Sweden AB,Stockholm, Sweden) to generate a stock solution of 100 mg/ml. In anEppendorf tube, 2 μl of cholesterol from the stock solution dilated in50 μl chloroform was added, subsequently ½ ml of water was layered onthe top of the cholesterol solution. The chloroform was evaporated by astream of air created with a syringe with a needle. A visible layer ofcholesterol was seen on the wall of the tube.

In Step 2

The ml of water was replaced by 1 ml of fresh water and 10 μl of the QHCstock solution (100 mg/ml in water) was added to the water, followed byincubation over night at 37° C. The membrane disappeared from the walland a clear water solution is seen.

-   -   A. Two μl of cholesterol stock solution and processed as        described step 1 and 10 μl of the QHC in step 2 were used to        generate this G3 formulation, which gave a molar ratio of 1:1    -   B. Another molar ratio was also used i.e. 2 mol of cholesterol        vs. 1 mol of QHC. Otherwise, the experiment was the same as for        A    -   C. A ratio of 1 mol cholesterol and 1 mol phosphatidylcholine        (P-5763 is from Sigma-Aldrich Sweden AB, Stockholm, Sweden) were        used to form the membrane in step 1. In step 2, two mol of QHC        was used.

Results

A. After evaporation, a 17 nm particle having a uniform size wasachieved characterized by electron microscopy (EM) see FIG. 1 A and bygradient centrifugation. The G3 suspension is visualized as a clearsolution.

B. After evaporation, particles of a slightly wider size range wereobserved in EM with a medium diameter of about 17 nm (not shown), i.e.17 nm particles were also created within the range of 2 ml ofcholesterol and 1 mol of Quillaja saponin.

C. The product had morphology like that of ISCOM particles with adiameter of about 40 nm (FIG. 1B) Thus, the morphology is completelydifferent with the inclusion of phosphatidylecholine from that of thenanoparticles according to the G3 invention without thephophatidylecholine. It can also be concluded that the nanoparticlesaccording to the invention is an excellent basis for integration ofphopholipids including phosphatidylecholine being essential for ISCOMformulation as claimed by Lövgren & Morein¹.

Conclusion and Discussion

With the molar ratio of 1 cholesterol to 1 Quillaja, small (17 nm)nanoparticle is formed. The higher ratio of cholesterol i.e. molar ratioof 2 cholesterol vs 1 Quillaja or more, then larger particles appear. Tonote, by inclusion of other lipophilic components in step 1, the size ofthe particle will vary, i.e. the loading of the particle influences thesize. In this case, the range of size was recorded between 17 and 40 nmbut that is not the limitation of the range. Especially important isthat in EM no aggregation of the particles was seen rather the particleswere well dispersed from each other.

An essential and new concept to reader lipophilic substances watersoluble is this two step procedure. There are various ways of forminglipid membrane e.g. liposomes that have no solid hydrophobic support,but are in free suspension in a water phase. The second step extractsthe membrane into the water phase via first into the water soluble G3particle.

To note, the procedure for forming nanoparticles according to theinvention is robust and much simpler than now used methods e.g. to makeISCOM formulations (Lövgren & Morein, see above). Above all, with theevaporation used them is hardly any loss of material used for theparticle formulation i.e. Quillaja saponin is the referred case QHC orcholesterol and no phospholipid is required, further reducing theproduction costs. Interestingly, the nanoparticles according to theinvention can be used as a base for forming ISCOMs.

If detergent is used for solubilisation the removal of detergent has tobe done as described by Lövgren & Morein² e.g. by dialyses, columnchromatography, ultracentrifugation or tangential flow it may be morepractically to use beads (e.g. latex beads) with a hydrophobic surface.Thus, the two step procedure is an innovative robust method toformulating nanoparticles, which may vary in morphology depending on theload.

Moreover, this method that in step 1 formulates a membrane is a generalmethod for incorporating lipophilic molecules into the water soluble G3particle in step 2.

Example 2

This example shows that a molecule with amphipathic properties can beincorporated into the G3 particles according to technology as describedin Example 1. To note G3 is naturally soluble in water as a micellebeing disintegrated after administration due to dilution at the site ofinjection and subsequently after the transportation from the site. Ithas therefore low poor bioavailability. Consequently a stable complex inG3 is important.

Experimental Set-Up

The experiment set-up is essentially the same as for Example 1, apartfrom that a hydrophobic molecule diterpenoid (DT) was also solubilizedat the same time with cholesterol as described for Example 1. The DTmolecule together with Quil A fraction C (QHC) and cholesterol wasincorporated in the G3 particle using the same two steps procedure as inexample 1 i.e. 1 μl DT (100 μg/ml in 99% ethanol as the stock solution)was solubilized in a molar ratio of 1 cholesterol:0.5 DT described forstep 1 in example 1. In step 2, QHC was added in to molar ratio of 1 to1 with cholesterol in example 1.

Results

The G3 particle with incorporated DT has the same morphology as the G3particle without DT depicted in FIG. 1A. In step 1 a membrane wasvisualized on the walls of the tube that disappeared in step 2. Thewater solution from step 2 is clear to slightly opalescent and nosediment was detected.

Conclusion

The amphipathic molecule diterpenoid (DT) in the micelle form has beensuccessfully disintegrated by the solvents used and in step 2 integratedinto the nanoparticles according to the invention, resulting in typicalG3nanoparticles of 17 nm. Thus, the capacity to use the G3 nanoparticlesaccording to the invention as a carrier/delivery system for anamphipathic molecules is shown. Amphipathic molecules with requiredconfiguration form micelles mostly instable after administration intoindividuals that often results in low bioavailability. In example 4 itis shown that this G3-DT particle is biology active. Further studieswill be performed during the Paris Convention priority year to furtherprove the immune enhancing capacity and usefulness of the nanoparticlesaccording to the invention as delivery particles and for enhancingbiological effects by interaction with cells e.g. via receptors in thecells membranes. For more information see Hu et al.³, incorporatedherein by reference. By incorporation of other molecules withcomplementary properties including the immune enhancing and the cancercell killing effects will potentially be substantially broadened.

Example 3

The capacity of G3 to kill cancer cells were tested in vitro on the U937model representing a lymphoid tumour cell.

Experimental Set-Up

G3 were formulated with various weight or molar ratios betweencholesterol and QHC as described in Materials and Methods. The variousformulations were incubated and the cancer cell killing effect wasmeasured after staining and reading by the FMCA method as described inMaterials and Methods.

Results

G3 particles formed with the ratio of 1 cholesterol vs 5 QHC accordingto the weight i.e. 1 cholesterol vs 1 QHC according to the molarity, asdescribed in Ex 1 and concluded from morphology according to the EMresults (see Ex 1 and FIG. 1A.). The U937 cancer cell killing effect ofG3 particle was of the same magnitude as for KGI particles (FIG. 2),indicating that the active component QHC was preserved when incorporatedin the G3 formulation. KGI has previously been known to kill U937 cancercells (PCT/SE 2005/050878).

Example 4

This example shows that the amphipathic molecule DT can be incorporatedinto the G3 particles according to technology as described in Examples 1and 2.

DT is a diterpen, a stevioside produced from Stevia rebaudianabertoni¹⁰. We have used DT because it has a number of interestingmedical including immunological properties as published¹¹. One problemwith this compound is that it has low bioavailability in vivo as we haveexperienced requiring a delivery system e.g. in a nanoparticle.

The capacity of DC to induce the cancer cell U937 to produce IL-8 wasdone to explore immunological effect but even more importantly todemonstrate with the cytokine that DC may lead the cancer todifferentiation being important to ceasing the uncontrolled cancer cellproliferation. Furthermore, the capacity of DT to induce human DCcytokines including IL-12 was tested. These tests were carried out toemphasize that DT has an important complementary immunological effect toQuil A and it is, therefore, useful to be incorporated in the G3particles. DT was supplied by Prodalysa LTDA, Santiago, Chile. Here weused human dendritic cells (DC) prepared as described in Material andMethods.

Experimental Set-Up

The U937 cells were incubated with KGI or G3 formulations starting from100 μg/ml followed by 5-fold dilutions for 6 steps for 48 hours at 37°C. Then the supnatant was collected and used for IL-8 detection asdescribed in Materials and Methods

For the measurement of gene expression, human DCs were incubated withBBE, KGI, Stevia and Stevia in combinations with BBE or KGI (FIG. 4A) aswell as G3 with or without Stevia incorporated (FIG. 4B) for 6 hours.The expression of various cytokine genes from the treated DCs wascarried out by mRNA array analysis as described in Materials andMethods.

Results

G3 particles induced U937 cancer cells to produce a similar level ofIL-8, which is a differentiation marker of the cancer cell, productionto that induced by KGI (FIG. 3).

FIG. 4A shows that DT induced high levels of IL-12, IL1β, and IL-6,higher than those induced by the ISCOM like formulations KGI and BBE

The G3 particles with DT incorporated induced high level of IL-12, whilethe G3 formulation without DT incorporated did not induce detectablelevel of IL-12 (FIG. 4B).

Discussion and Conclusion

DT has complementary properties to G3. The capacity to induce the U937cancer cells to produce IL-8 may indicate immune enhancement. Moreimportantly, G3 standalone can differentiate and lead the cancer cell tocease proliferation like KGI as we have shown (manuscript in preparationto be supplemented). Complementary effect to G3 alone it is shown thatDT potently induces IL-12, which is important for induction of a Th1type of response particularly for production of IFN-, which hasanticancer effect for certain tumors. Foam immunological point of view,IL-12 is important for rejection of tumors if there are tumor antigensrecognized by the immune system. IL-12 is also important for the immunedefense against virus infections. It is of particular interest to recordthat DT is incorporated into G3 serving as a carrier/delivery particle,which increases its stability in a particle form after administrationand consequently increases G3's bioavailability. To note, DT stand aloneis in a micelle form in water. A micelle formation administered into thebody of an individual disintegrates because the dilution at the site ofadministration and subsequently decreases as it is transported from thesite. The G3 particle is held together by other forces and does notdisintegrate e.g. after injection that has been recorded by EM studies(not published). This example emphasizes that the G3 invention serves ascarrier for amiphipathic molecules.

Example 5

Inhibition of thymidine kinase (TK) activity by G3 particles is anessential property to prevent uncontrolled replication of cancer cellsand for the subsequent steps to steer the cell to a programmed celldeath i.e. apoptosis.

To explore one mechanism of G3 particle to inhibiting the cancer cellreplication was evaluated by measuring the inhibition of TK enzymatic onU937 cells. Besides using inhibition of the TK enzyme activity forshowing inhibition of cancer cell replication this mode of action willbe used to evaluate whether G3 particles or any other quillajaformulation used for anticancer treatment will be useful to treat tumourpatients stand alone or in combination with other anticancerformulations. Such a test can be used in a kit to measure thesensitivity of anticancer drugs using inhibition of TK mode of action onclinical samples in the field defined as personalized medicine.

Experimental Lay-Out

U937 cells were exposed to the same amounts of G3 or KGI. At varioustime points, the treated cells were collected and inter-cellular TKactivities were measured as described in Materials and Methods.

Results

G3 particles inhibit virtually the same magnitude of inter-cellular TKactivities as that by KGI (FIG. 5).

Conclusion

The inhibition of TK activity shows that the cancer cell ceases toreplicate. The inhibition of TK activity on the cellular level cantherefore be used to measure the sensitivity of cancer cell from thepatient samples to the drug i.e. the G3 particles, paving the way topersonalized medicine. To our knowledge, TK activity has been used as anon-specific test for the detection of serum TK from cancer patients orother disease indications from patients: By using it directly on cancercells from patients is our innovation (with 100% specificity i.e. onlycancer cells are used). This, together with the cancer cell killingproperty, makes G3 particles more feasible for clinical use by avoidingits use on non-responding patients.

Part II. G3 as an Anticancer Drug Example 6

This example shows G3 and G3 with DT (G3-DT) kills the non-solid tumorhuman Acute Myeloid Leukemia (AML) cells more efficiently than theactive component QHC (QHC) in G3 in a non-particulate form.

Experimental Set-Up

The nanoparticles G3 and G3DT formulated as described in Ex 1 and 2 werecompared with the active component QHC form for the cancer cell killingeffect. The samples were 5-fold serial diluted in 6 steps starting from100 μg/ml, and incubated or 3 days with HL-60 AML cells f. Then thecells were stained and read by the FMCA method.

Result

G3 (IC₅₀=3.144 μg/ml) and G3-DT (IC₅₀=3.12 μg/ml) inhibited the growthof the AML cells more efficiently than QHC (IC50=8.473 μg/ml) (FIG. 6).

Conclusions and Discussions

Essentially, G3 has a stronger cancer cell killing effect compared tothat of the non-particulate QHC. Incorporating DT into the G3 particlesforming G3-DT enhances the cancer cell killing effect compared to eitherQHC or G3 without DT. DT alone has no cancer cell killing effect (notshown). The non-particulate QHC causes local reaction that is abolishedby the particulate forms. The added effect of G3-DT in killing AMLcancer cells would also implicate a beneficial dose reduction.

Example 7

Cytarabine is a commercially available cytostatic drug used fortreatment of Acute Myeloid Leukemia (AML). This example was set up toexplore the capacity of G3 to enhance the cancer cell killing effect ofcytarabine.

Experimental Set-Up

HL-60 AML cells were exposed for 3 days at pre-determined concentrationsof G3 and cytarabine separately and in combination of these two as shownon FIG. 7. Then the cells were stained and read for cancer cell killingeffect by the FMCA method.

Result

After incubation for 3 days, G3 or cytarabine alone killed less than 5%and 55% the cells respectively. When they were combined, the killingrate was elevated significantly (P<0.01) to about 75% (FIG. 7).

Conclusions and Discussions

The G3 particles significantly enhance the killing effect of cytarabineon HL-60 AML cells. Treatment with the cytostatic drug cytarabine causesside effects with discomfort for patients. Since G3 particles arevirtually non-toxic, the combination treatment with G3 and cytarabinewould also have prospect for increasing efficacy and reduce the sideeffect by lowering the dose of cytarabine.

Example 8

This example demonstrates that G3 has added cancer cell killing effecton the commercial cytostatic cancer drug daunorubicin on the non-solidtumor human Acute Myeloid Leukemia (AML) cells.

Experimental Set-Up

HL-60 AML cells were exposed to a fixed concentration (1 μM) of G3combined with increasing concentrations of daunorubicin starting from1000 nM, and incubated for 3 days. Then the cells were stained and readby the FMCA method.

Result

G3 enhances significantly (P<0.0001) the cancer cell killing effect ofdaunorubicin compared to daunorubicin stand alone (FIG. 8).

Conclusions and Discussions

The G3 particles enhance synergistically the killing effect ofdaunorubicin on HL-60 AML cells. Since G3 particles are virtuallynon-toxic, it is likely the dose of the cytostatic drug daunorubicinwould be considerably reduced in a combination therapy with G3implicating better treatment effect, and because of lowered side effectthe treatment can be continued for longer periods in patient sensitiveto daunorubicin.

Example 9

This example was designed to compare the effects of G3 and G3 with DTincorporated (G3DT) on solid tumors exemplified by human prostate cancercells PC-3.

Experimental Set-Up

G3 and G3DT was compared with non-particulate QHC. The samples were5-fold serially diluted in 6 steps starting from the concentration of100 μg/ml, and incubated with PC-3 prostate cancer cells for 3 days.Then the cells were stained and read by the FMCA method.

Result

The G3 particles (IC₅₀=2.75 μg/ml), and the G3-DT (IC₅₀=1.662 μg/ml)incorporated inhibited the growth of the prostate cancer cells morepotently than the same active component QHC alone (IC₅₀=3.388 μg/ml)(FIG. 9)

Conclusions and Discussions

Essentially, G3 in this example had a similar killing effect on the PC-3prostate cancer cells as that of QHC. By incorporation of DT into theparticle i.e. G3-DT, the cancer cell killing effect of G3 is, as withAML cells, enhanced. It is known that DT alone has no cancer cellkilling effect. Its added effect here in killing PC-3 cancer cellsimplicates enhancement of the G3 effect and also reduction of the sideeffects if any. To note the QHC in non-particulate form is comparativelyefficient in vitro but in vivo QHC remains at the site of injectionresulting in low bioavailability and local side effects.

Example 10

This example demonstrates that the combination effect between G3 and acommercial drug docetaxel on solid tumors exemplified by PC-3 prostatecancer cells.

Experimental Set-Up

PC-3 cells were exposed for 3 days at pre-determined concentrations ofG3 and docetaxel separately and in combination as shown on the graph.Then the cells were stained and read by the FMCA method.

Result

The cancer cell killing effect of G3 or docetaxel alone killed slightlymore than 35% and 2% the cells respectively. When these two drugs werecombined, the killing rate was significantly elevated (P<0.01) to about75%. (FIG. 10).

Conclusions and Discussions

The G3 particles significantly enhance the cancer cell killing effect ofthe cytostatic docetaxel on the prostate PC-3 cancer cells implicatingincreased efficacy and reduced dosing of the cytostatic drug withreduced side effect in view of the fact that G3 particles are virtuallynon-toxic.

Example 11

This example was designed to explore combination effect between G3 and arecent and under patent covered cytostatic commercial drug cabazitaxelon the solid tumor human prostate cancer PC-3 cells.

Experimental Set-Up

PC-3 prostate cancer cells were exposed to a fixed concentration (1 μM)of G3 combined with increasing concentrations of cabazitaxel startingfrom 100 μM, and incubated for 3 days. Then the cells were stained andread by the FMCA method.

Result

Cabazitaxel alone killed the PC-3 cancer cells at, G3 at an IC₅₀=25.54μM. Cabazitaxel combined with G3 the cancer cell killing effect(IC₅₀=0.00023 μM) was significantly (P<0001) enhanced (FIG. 11).

Conclusions and Discussions

The G3 particles significantly and synergistically enhance the killingeffect of cabazitaxel on PC-3 prostate cancer cells implicatingprospects for better efficacy, reduced side effect and possibility forprolonged treatment in view of the fact that G3 particles are virtuallynon-toxic.

Example 12

This example was designed to explore the capacity of G3 particlesformulated with another important Quillaja saponin fraction QHA inkilling solid tumours exemplified here with ACHN kidney cancer cell linesince it was observed before that Duecom particles formulated with thisfraction had a stronger killing capacity than Deucom particlesformulated with Fraction C (QHC).

Experimental Lay-Out

G3 formulated with QHA and QHC were diluted 5-folds, 6 steps from 100μg/ml down to 0.032 μg/ml and incubated with ACHN renal carcinoma cellsat 37° C. for 3 days. The cell survival was determined by the FMCAmethod.

Result

G3 formulated with QHA kills significantly (P<0.01) more ACHN kidneycancer cells than that of G3 formulated with QHC (FIG. 12).

Conclusions and Discussions

This result is virtually identical to the previous observation withDeucom particles formulated with QHA and QHC i.e. that formulations withQHC is selectively killing more non-solid tumour cells whileformulations with QHA is preferably killing more solid than non-solidtumours. This tumour type specific killing property with G3 formulationscould be harnessed as for Deucom particles to avoid killing normal cellsin another category.

Part III. G3 as Adjuvant Example 13 Animal Trial of G3 Particle as anAdjuvant Against Whole Virus

The adjuvant effect of G3 in comparison to ISCOMs was evaluated in ananimal trial on C57BL/6 mice. The disintegrated and inactivatedinfluenza virus was used as the model antigen in the experiment.

Experimental Lay-Out

Six mice per group, immunized twice 4 weeks apart, blood samples weretaken at 3 weeks after the first immunization and 4 weeks after thesecond immunization (see the graphic description on next page). At thenecropsy i.e. 4 week after the second immunization, spleen cells wereanalyzed for cytokine production as described in materials and methods.To facilitate the understanding, grouping of the animals is shown inFIG. 13A.

Result

-   -   The G3 and ISCOM induced in dose dependent manner detectable        levels of HI antibody after the 1^(st) immunization. After the        2^(nd) immunization, the level of HI antibody increased        considerably (a clear boost effect) also in a dose dependent        manner for the G3 adjuvanted formulations. The ISCOM, G3 and G3        with DT incorporated adjuvanted formulations induced        considerably higher levels of III antibody than the        non-adjuvanted commercial vaccine, i.e. similar or higher levels        of HI responses were recorded between animals immunized with G3        and ISCOM formulations at two time points after the 1^(st) (FIG.        13A) and the 2^(nd) (FIG. 13B) immunizations.    -   Similar or even higher levels of IFN-γ and IL-4 responses were        detected in spleen cells after in vitro re-stimulation with the        split virus between animals immunized with G3 and ISCOM        formulations at the necropsy, 4 weeks after the 2^(nd)        immunization (FIG. 13C).

Conclusion

Both antibody-mediated and cell-mediated immunities induced by G3 andISCOM formulations are qualitatively and qualitatively the same, i.e. G3formulations i.e. G3 and G3DT can replace the ISCOM as adjuvant.

Part IV. G3 as a Drug Delivery System Example 14

VLX40 was considered to be a promising drug for treating cancer. Theresearch to market ceased because VLX40 could not be formulated to besuitable for administration to animals in preclinical tests andconsequently not for subsequent tests in patients. The reason was thatVLX40 could not be rendered soluble in water, which is necessary to betaken up by the body. This experiment was designed to find out if the G3technology could solve the problem and make VLX40 water soluble.

Experimental Set-Up

-   -   VLX 40 was first dissolved in an organic solvent DMSO to yield a        concentration of 20 mM (5.8664 mg/ml, the highest concentration        recommended) and designed as the stock solution.    -   VLX40 at a concentration of 100 □g/ml was used as a control for        VLX40 in water i.e. as a virtually non-water soluble        formulation.    -   8.5 μl of the VLX40 stock solution was mixed with 50 μl        chloroform in an Ependorf tub containing 500 μl water to form an        artificial lipid membrane (see Example 1). In the second step,        the 10 μl Quil A (100 mg/ml in water as the stock solution) was        added and incubated overnight at 37° C. This is the G3-VLX40        formulation, essentially formed in the same way as described in        Example 1.    -   The VLX40-DMSO: control (2) and the G3-VLX40        suspension/solution (3) were collected and tested on U937-GTB        cells after being serially diluted from the same concentration        as above, and IC50s were calculated from the regression curves.

Results

The stock solution (1) added to water gave rise to a visible sediment orprecipitate i.e. as expected from previous experiments. Thus, VLX40could not be dissolved in water from the control tube (2). Theformulation of G3-VLX40 particles (3) was confirmed by electronmicroscopy of the clear solution/suspension. There was no precipitate oronly very scanty precipitate from the tube containing the G3-VLX40formulation.

The various formulations were tested for function i.e. in bioassay forthe cancer cell killing effect of U937 cells (see Materials andMethods). The water phase of the VLX40/DMS0 (2) had low anticancereffect (expressed as IC 50), meaning that just a very small fraction ofVLX 40/DMSO mixture is dissolved in water (FIG. 14A).

In contrast, the water phase of the G3 VLX-40 particle formulation (3)had high anticancer cell activity as shown in FIG. 14A.

The sediment non-soluble part was also tested in vitro for the cancercell killing effect. High cancer cell killing effect was recorded withVLX 40/DMSO (2) in the water-insoluble precipitate, the scantywater-insoluble precipitate of G3-VLX 40-formulation had a low cancercell killing effect (FIG. 14B).

These experiment has been repeated 3 times.

Conclusion and Discussion

About 50-100 micrograms VLX 40 was dissolved with 2 mg G3 measured asthe Quillaja component in a volume of 1 ml of water. It should be notedthat the concentration of G3 particles can be increased by increasingthe concentration of the G3 particles, for example 10 mg/ml of the G3formulation can be in a clear solution. We can also change thecomposition of the G3 particles to facilitate the incorporation oflarger amount VLX 40. This example clearly demonstrates that the G3particle is filling an unsolved demand to facilitate formulation ofdrugs for clinical use that without the G3 technology could not reachthe patient because the drugs could not be made water soluble with theexisting technologies.

Example 15

This example demonstrates that G3 particle, as a drug delivery system,can incorporate readily another two non-water soluble anticancer drugsbusulfan and roscovitine, making them water soluble.

Experimental Set-Up

Two μl busulfan (50 mg/ml in DMSO) or 1 μl roscovitine (100 mg/ml inchloroform) together with 2 μl cholesterol (100 mg/ml in chloroform)were used to form the lipid membrane with busulfan or rocovitinerespectively, using the way method as described for step 1 in Example 1.Then 10 μl QHC (100 mg/ml in water) was added as for step 2 in Example 1to give a molar ratio of QHC:cholesterol:busulfan/roscovitine=1:1:0.5.

Result

For both compounds i.e. busulfan and roscovitine, clear solutions werevisualized i.e. sediment or cloudiness in the water phase caused by theinsoluble drugs were eliminated by incorporating them into the watersoluble G3 particles.

Discussion

This example, similar to example 13, shows once again that the capacityof G3 as a general platform for making non-water soluble lipiphilicdrugs/molecules water soluble by incorporating them into the G3particles. Considering that about 40% of the anticancer drug candidatesare not water soluble, therefore, cannot be further developed intocommercial products, our invention can drastically improve thesituation.

Example 16

In this example, we have explored whether a lipophilic vitamin i.e.vitamin D3 can be integrated into the G3 nanoparticle in order to makeit water soluble. It was dissolved in chloroform and incorporated intothe G3 particle as described in example 1 and for more details seeMaterials and Methods.

Experimental Set-Up

G3 particles were formed using 50% cholesterol and 50% vitamin D3 toform the lipid membrane. Quil A was added into the water phase togenerate the G3 particles (for details, please refer to Example 1). 100%Cholesterol and 100% Vitamin D3 in water were used as the controls.Samples with vitamin D3 incorporated in G3 particles were analysed atthe Uppsala University Hospital Laboratory on a DiaSorin Liaisonautomatic instrument.

Result

The water phase recovered in step 2 was a clear solution and no sedimentcould be detected. More vitamin D3 was detected in the G3-vitamin D3formulation based on non-fractionated quillaja (550 nmol/L) than in thequillaja QHC fraction formulation i.e. 55 nmol/L. In a dilutionexperiment the concentration of vitamin D3 was linear in the read outshowing that them was a homogenous suspension of particles i.e. noaggregation being in agreement with other G3 particles as seen inFIG. 1. In comparison, only trace amount of Vitamin D3 was detected inthe vitamin D3 control and no Vitamin D3 was present in the cholesterolcontrol.

Conclusion and Discussion

Vitamin D3 is an essential vitamin that is poorly taken up by the bodyin the lipid form by oral or parenteral routes. Thus, a water solubleform will facilitate its uptake by those routes. We show in thisexperiment that vitamin D3 is incorporated into the G3 nanoparticle withthe non-fractionated as well QHC fraction of quillaja saponin.Importantly, the linear read out of the dilution experiment shows ahomogenous dispergation of the particles that has been revealed byelectrone microscopy for G3 particles in general (see FIG. 1) For foodthe non-fractionated quillaja saponin is well accepted and used e.g. inbeverages including bear and also other types of food. Therefore, themore economical alternative for the formulation of G3 for delivery ofthis vitamin is a non-fractionated quillaja as base for the G3formulation. In this experiment more D3 was incorporated into the G3with non-fractionated quillaja saponin than in the G3 particle with theQHC saponin fraction.

REFERENCES

¹ Kefei Hu, Saideh Berenjian, Rolf Larsson, Joachim Gullbo, PeterNygren, Tanja Lövgren, Bror Morein Nanoparticulate Quillaja saponininduces apoptosis is human leukemia cell liens with a high therapeuticindex. International Journal of Nanomedicine, January 2010 Volume 2010:5Pages 51-62

² Lövgren & Morein (2000) ISCOM Technology in Methods in MolecularMedicine, Vol 42:239-258, Vaccine adjuvants: Preparation Methods andResearch Protocols, Edited by D. T. O O'Hagen, Humana Press, Inc.,Titawa, N.J.

³ Morein B, Hu K, Lovgren K and D'Hondt E, New ISCOMs meet unsettledvaccine demands in Vaccine Adjuvants and Delivery Systems, Ed. by SinghM. A John Wiley & Sons, Inc., Publication, Hoboken, N.J. 2007, p191-222.

⁴ Lycke, N, From toxin to adjuvant: The rational design of a vaccineadjuvant vector, CTA1-DD/ISCOM. Cell Microbiol 2004, 6(1), 23-32, and byMowat et al. (2001)

⁵ Mowat, A. M., Donachie, A. M., Jagewall I. S., Schon, K., Lowenadler,B., Dalsgaard, K., et al CTA1-DD-immune stimulating complex: a novel,rationally designed combined mucosal vaccine adjuvant elective withnanogram doses of antigen. J. Immunol 2001, 167(6), 3398-3405

⁶ Blair A H, Ghose T I. Linkage of cytotoxic agents to immunoglobulins.J Immunol Methods, 1983 Apr. 29; 59(2):129-43.

⁷ Ghose T I, Blair A H, Kulkarni P N, Preparation of antibody-linkedcytotoxic agents. Methods Enzymol. 1983; 93:280-333.

⁸ Davis M T, Preston J F. A simple modified carbodiimide method forconjugation of small-molecular-weight compounds to immunoglobulin G withminimal protein crosslinking. Anal Biochem. 1981 Sep. 15; 116(2):402-7.

⁹ Eliasson D G, El Bakkouri K, Schön K, Ramne A, Festjens E, LöwenadlerB, Fiers W, Saelens X, Lycke N, CTA1-M2e-DD: a novel mucosal adjuvanttargeting influenza vaccine. Vaccine, 2008 Feb. 26; 26(9):1243-52. Epub2008 Jan. 10.

¹⁰ A. Esmat Abou-Arab, Azza Abou-Arab and M. Ferial Abu-Salem,Physico-chemical assessment of natural sweeteners steviosides producedfrom Stevia rebaudiana bertoni, African Journal of Food Science, Vol 4(5) pp 269-281, May 2010.

¹¹ Chaiwat Boonkaewwant, Chaivat Toskulkao, and Molvibha Vongsakul.Anti-Inflammatory and Immunomodulatory Activities of Stevioside and ItsMetabolite Steviol on THP-1 Cells, J. Agric. Food Chem. 2006, 54, 7857897)

¹³ Kersten, G. F. A., Spiekstra, A., Beuvery, E. C. and Cromelin D. J.A. (1991) On the structure of immune-stimulating saponin lipid complexes(ISCOMs), BBA 1602, 165-171.

1. Nanoparticles comprising sterol, preferably cholesterol and acomponent from Quillaja saponaria Molina selected from quillaja acid andquillaja saponin, characterized in that said nanoparticles do notcomprise a phospholipid.
 2. Nanoparticles according to claim 1, having aparticle diameter in the range of 10-40 nanometers, preferably 12-35nanometers or 15-25 nanometers.
 3. Nanoparticles according to claim 1 or2, wherein said quillaja component is a quillaja saponin, preferablyquillaja saponin fraction QHA, QHB and/or QHC.
 4. Nanoparticlesaccording to any of the preceding claims, wherein the ratio betweencholesterol and quillaja saponin is from 1:10 to 10:1, preferably from1:2 to 2:1.
 5. Nanoparticles according to any of the preceding claims,further comprising at least one an amphipathic or a hydrophobicmolecule.
 6. Nanoparticles according to claim, 5, wherein theamphipathic or hydrophobic molecule is at least one member selected froman antigen, an adjuvant, a targeting molecule, a pharmaceutical compoundand a food related compound.
 7. A composition comprising one or morenanoparticles according to any of claims 1-6.
 8. The compositionaccording to claim 7, wherein different quillaja saponin fractions areeach incorporated in different nanoparticles.
 9. A nanoparticleaccording to any of claim 1-6 or a composition according to any ofclaims 7 and 8 for use as a pharmaceutical optionally in apharmaceutical composition further comprising pharmaceuticallyacceptable buffers, diluents excipients, adjuvants and/or carriers. 10.A pharmaceutical composition according to claim 9, further comprising atleast one pharmaceutically active compound, such as anticancer drugsKomplettering Saideh, platinum coordination compounds, taxane compounds,camptothecin compounds, anti-tumour vinca alkaloids, anti-tumournucleoside derivatives, nitrogen mustard or nitrosourea alkylatingagents, anti-tumour anthracycline derivatives, trastzumab andanti-tumour podophyllotoxin derivatives, antimetabolites, Steroids,inhibitor of mammalian target of rapamycin (mTOR), agents for treatingcancer, such as agents selected from the group consisting of Cytarabin,Daunorubicin, Paclitaxel, Docetaxel, Cabazitaxel, Toricsel andTrabectidin, which active compound may be integrated into thenanoparticle or mixed with the composition. Quillaja saponaria Molinaand sub fragments thereof, receptors for antibodies or monoclonalantibodies such as Fc receptors or the DD of Protein A of Staphylococcusaureus.
 11. The pharmaceutical composition according to claim 9 for useas an adjuvant.
 12. A pharmaceutical adjuvant formulation according toclaim 11 for use in combination with a vaccine under development
 13. Thepharmaceutical adjuvant formulation according to claim 11, for use incombination with a seasonal influenza virus vaccine
 14. Thepharmaceutical adjuvant formulation according to any of claims 11-13,for use in combination with a pandemic influenza vaccine
 15. Thepharmaceutical adjuvant formulation according to any of claims 11-14,for use in combination with an emergency vaccine, such as a vaccineagainst a biological weapon.
 16. Pharmaceutical vaccine formulationcomprising an adjuvant according to any of claims 11-15.
 17. A methodfor treating or preventing a disease caused or complicated by anorganism, comprising administering to a subject a pharmaceutical vaccineformulation according to claim
 16. 18. A method for treatment of cancer,comprising administering to a patient in need thereof a pharmaceuticallyeffective amount of nanoparticles according to any of claims 1-6 or acomposition according to claims 7-10.
 19. The method for treatment ofcancer according to claim 18, wherein said cancer is leukemia.
 20. Amethod for producing phospholipid-free nanoparticles comprising thesteps a) providing a hydrophobic surface or a suspension of liposomes b)bringing hydrophobic surface or the suspension of liposomes into contactwith a solution of sterol, preferably cholesterol dissolved as monomersin an organic solvent or detergent; c) removing the solvent or detergentforming a sterol membrane on the surface d) providing a water solutionof quillaja saponin micelles e) adding the water solution comprising thesaponin micelles to the sterol membrane, whereby a complex is formedbetween the saponines and the sterols and is suspended in the watersolution.
 21. The method according to claim 20, wherein the organicsolvent is ethanol and/or chloroform.
 22. The method according to any ofclaims 20 or 21, wherein the organic solvent or detergent is removed byevaporation.
 23. The method according to any of claims 20 or 21, whereinsaid solvent or detergents are removed by dialysis, chromatography,filtration or tangential flow.
 24. The method according to any of claims19-22, wherein said quillaja saponin is quillaja saponin fraction QHC.25. The method according to any of claims 19-24, wherein the ratiobetween cholesterol and quillaja saponin is from 1:10 to 10:1,preferably from 1:2 to 2:1.
 26. A method for production of Iscom matrixaccording to any of claim 20-25, wherein at least one phospholipide isadded to the suspension comprising sterol in step b) in claim
 20. 27.Method for assessing the applicability of a method according to claim 17or 18 to an individual patient, comprising bringing cancer cells fromsaid patient in contact in vitro with nanoparticles according to any oneof claims 1-7 or a pharmaceutical composition according to claim 8 or 9;measuring at least one effect indicative of therapeutic effect of saidnanoparticles or pharmaceutical composition on said cancer cells;wherein the method according to claim 10 or 11 is assessed as applicableto said individual patient if the nanoparticles or pharmaceuticalcomposition shows a significant effect indicative of therapeutic effecton said cancer cells.
 28. The method according to claim 27, wherein theeffect indicative of therapeutic effect is down regulation of cyclinedependent kinases (CDKs) or down regulation of thymindine kinase (TK) upregulation of differentiation markers including.